Fruit ferments containing propionate and use thereof

ABSTRACT

The present invention discloses a method to prepare a fruit ferment containing propionic acid and/or a salt thereof comprising i) obtaining a liquid fruit preparation, ii) optionally supplementing the liquid fruit preparation with water and/or with additional components to support fermentation, iii) fermenting the optionally supplemented liquid fruit preparation with a propionic acid-producing bacterial strain, iv) optionally further processing the fermentation product, to obtain the fruit ferment. Next to a propionic acid-producing bacterial strain, a lactic acid-producing bacterial strain may be used in the fermentation. The invention also relates to a fruit ferment obtainable by said method and the use in a food, beverage, as a probiotic and to improve the sensory properties and/or the storage stability is also disclosed.

The present invention relates to fruit based fermented productscontaining propionate and uses thereof.

Yeasts and acetic acid bacteria are the most common microorganismsinvolved in the spontaneous fermentation of fruits, convertingcarbohydrates to alcohol and/or acetic acid. Also, these organisms canact as spoilage organisms of fruit based products such as juices. On theother hand, Propionibacteria are involved in the fermentation of dairyproducts, most commonly cheeses, converting carbohydrates and lacticacid to mainly propionic acid, but also some acetic acid, carbon dioxideand succinic acid. Propionic acid especially inhibits yeasts and mouldsbut also other bacteria.

Babuchowski et al. (Lait (1999), 79, 113-124) discloses the use ofPropionibacteria to produce fermented vegetables. The propionic acidcontent of red beet juice fermented with Propionibacteriumfreudenreichii and Propionibacterium thoenii, respectively, reached avalue of 1.56% and 2.18%, respectively, after 14 days of fermentation.Such a fermentation period is much to long for industrial application(high costs and high risk for contamination). When fermentation was donewith propionibacteria and lactic acid bacteria together, the propionicacid content of the fermented red beet juice reached a maximum value ofonly 0.12% after 1 day of fermentation, whereas prolonged fermentationof 10-14 days resulted in a lower propionic acid content (about 0.04%).

It was now surprisingly found that when liquid fruit preparations, likejuices, extracts, purées, were purposively fermented with members of thegenus Propionibacterium, a ferment can be obtained having a highpropionate content, even without the addition of additional nutrients,when fermentation was done for a period of 1 to 4 days. The fermentinhibits yeasts and moulds when applied in e.g. fruit based products,but, advantageously, hardly influences the taste and flavour of thefruit based product, even when applied in a high concentration.

Thus, in a first aspect the present invention discloses a method toprepare a fruit ferment containing propionate by subjecting a liquidfruit preparation to fermentation by a propionic acid-producingbacterial strain. The method in particular comprises:

i) obtaining a liquid fruit preparation,

ii) optionally supplementing the liquid fruit preparation with waterand/or with additional components to support fermentation,

iii) fermenting the optionally supplemented fruit preparation with apropionic acid-producing bacterial strain,

iv) optionally further processing the fermentation product, to obtainthe fruit ferment.

A fruit for use in the methods and fruit ferments as described herein isthe edible, fleshy, seed-associated structure of certain plants. Such afleshy structure typically has a natural water content of at least 65%(w/w), even at least 70% (w/w) or at least 75% (w/w). Such fruitsinclude sweet fruits like apples, pears, oranges, grapefruits, grapes,strawberries, melons, bananas, pineapples, passion fruits, peaches,mangos, guavas, non-sweet fruits like lemons, avocados, andvegetable-like fruits like tomatoes, cucumbers, bell peppers, pumpkins.A preferred fruit for use in the method of this aspect is a sweet fruitand/or a tomato. An especially preferred fruit is apple, pear, orange,grape, pineapple, melon or tomato, or any mixture thereof. The fruitsfor use in the methods and fruit ferments as described herein do notinclude nuts and cereal grains.

The fruit of choice is processed to obtain a liquid fruit preparation.Suitable liquid fruit preparations for instance are a juice, an extractor a purée (mashed fruit). Processing of the fruit is done usingtechniques commonly known to those skilled in the art. The liquid fruitpreparation may be obtained from a concentrate. A single fruit type ormixture of two or more fruit types may be used.

The liquid fruit preparation optionally may be supplemented with waterand/or with additional components necessary to support and/or improvebacterial fermentation. This supplementation may be done prior to and/orduring fermentation.

Dilution of the liquid fruit preparation with water suitably may be doneto obtain a suitable sugar level for the bacterial strain used in thefermentation and to advantageously accelerate the fermentation process.A suitable sugar level of the liquid fruit preparation may be 1.5 to 10%(w/w).

The liquid fruit preparation, optionally diluted, may further besupplemented with additional components necessary to support and/orimprove bacterial fermentation. For instance, the additional componentsmay be additional carbon source such as glucose or sucrose, additionalnitrogen source such as yeast extract and/or additional minerals and/orvitamins.

Advantageously, the amount of additional components in general is low,so that sensory properties of the resulting ferment are not negativelyaffected. It is preferred not to supplement the liquid fruit preparationat all.

Fermentation is done using a propionic acid-producing bacterial strain.Preferred propionic acid-producing bacterial strains are strains of thegenus Propionibacterium. Suitable species include Propionibacteriumfreudenreichii, Propionibacterium shermanii, Propionibacteriumacidi-propionici, Propionibacterium thoenii and/or Propionibacteriumjensenii. Examples of other propionic acid-producing bacterial speciesare Clostridium propionicum, Selenomonas ruminantum and/or Bacteroidesruminicola and/or species of the genus Veillonella.

Preferred Propionibacterium species are Propionibacteriumfreudenreichii, Propionibacterium shermanii and/or Propionibacteriumacidi-propionici. A single strain or a mixture of two or more strains orspecies may be used.

In one embodiment, the fermentation with a propionic acid-producingbacterial strain is supplemented with one or more strains of lacticacid-producing bacteria. Suitable lactic acid producing-bacterialstrains are found within lactic acid bacteria belonging to the orderLactobacillales or within moderately thermophilic Bacillus species.Examples of lactic acid bacteria are Lactobacillus casei, Lactobacillusacidophilus, Lactobacillus delbrueckii, Lactobacillus paracasei,Lactobacillus helveticus, Lactococcus lactis and/or Lactococcusplantarum. Examples of moderately thermophilic Bacillus species areBacillus coagulans, Bacillus smithii and/or Bacillus thermoamylovorans.Moderately thermophilic Bacillus species are defined as Bacillus speciesthat are capable of growing at temperatures between 30-65° C.

Lactic acid-producing bacteria are advantageously used to convert sugarsin the liquid fruit preparation to lactic acid, which lactic acid isparticularly suitable as a carbon source for the propionicacid-producing bacterial strain. Supplementing the fermentation with oneor more strains of lactic acid producing bacteria may be done in variousways. It may be done by firstly fermenting the liquid food preparationwith one or more strains of lactic acid producing bacteria and thenfermenting the resulting lactic acid containing fermentation broth orfermentation fluid (i.e. with or without lactic acid producing bacteria)with a propionic acid-producing bacterial strain. It also may be done bysimultaneously fermenting the liquid food preparation with one or morestrains of lactic acid-producing bacteria and of propionicacid-producing bacteria. In the latter option, the lactic acid-producingbacteria may constitute, at the end of the fermentation process, from10% to 90% of the total weight of bacteria used in the fermentationprocess.

The fermentation process further preferably is substantially devoid ofcontaminating microorganisms, i.e. other microorganisms than the onesdescribed hereinabove. For instance, at the end of fermentation thepercentage of contaminating microorganisms is at the most 5% (w/w) ofthe total amount of biomass produced.

The fermentation conditions are typically chosen conform the growthrequirements of the microbial strain(s) to be used in the fermentationprocess.

It is important to maintain the pH of the liquid fruit preparation,initially and during fermentation, at a value between 4.5 and 7,preferably between 5 and 7. This may be done by the addition of asuitable base, e.g. sodium hydroxide, potassium hydroxide, calciumhydroxide, magnesium hydroxide, calcium carbonate, ammonium hydroxide,ammonium carbonate, and/or natural sources containing these alkalisubstances like lime, limestone, chalk.

The fermentation process is typically done for a time period suitable toconsume the majority of the available sugars, preferably all of thesugars, and preferably also the majority or all of the lactate, ifpresent. However, it is also possible to ferment only part of theavailable sugars. The available sugars typically are the sugarsnaturally present in the fruit and any sugar added as additional carbonsource. At the end of fermentation, the propionate level of thefermentation fluid (i.e. measured after separation of the microbialbiomass) may vary from 0.25 to 10% (w/w).

Typically, fermentation is carried out for a time period from 20 to 120hours, preferably from 20 to 100 hours, more preferably from 30 to 90hours.

The fermentation process is generally carried out at a temperature inthe range of 10 to 70° C., in particular in the range of 25 to 55° C.,more in particular in the range of 25 to 35° C.

After fermentation is ended, the fermentation product may optionally befurther processed as desired, e.g. depending on its intended use.Advantageously, it is in general not necessary to apply furtherprocessing steps to improve sensory properties.

For instance, solids including biomass may be separated from thefermentation product to obtain a fermentation fluid. Any method known tothe skilled person for this solid removal may be used, e.g.ultrafiltration, microfiltration, static decantation, or centrifugation.It is also possible to remove only part of the solids from thefermentation product.

The fermentation product including solids or the fermentation fluid fromwhich solids are separated may also be concentrated. Concentration maybe carried out by any method known in the art. It may be carried out toform a concentrated solution, for instance by concentrating up to about20 times, such as 8 to 16 times. Suitable methods include for example(vacuum) evaporation and methods based on membrane technology, such asreverse osmosis. It is also possible to prepare a solid granularproduct, via for example extrusion or spray-drying.

The fermentation product including solids, the fermentation fluid fromwhich solids are separated or the concentrated product may also bepasteurized or sterilized.

Standardization may be necessary to achieve a fruit ferment having aconsistent composition.

In a second aspect, the present invention relates to a fruit fermentcontaining propionate.

The fruit ferment of this aspect is obtainable by subjecting a liquidfruit preparation as described in the first aspect to fermentation usinga propionic acid-producing bacterial strain, optionally together with alactic acid-producing bacterial strain, as also described in the firstaspect.

The fruit ferment comprises propionate in a concentration ranging from0.25 to 80% (w/w), preferably from 0.5 to 70% (w/w), more preferablyfrom 1 to 60% (w/w). The propionate content thereby depends on the watercontent of the fruit ferment and on the presence or absence of bacterialbiomass. Typically, the fruit ferment may have a water content of 0 to99.5% (w/w), depending on the further processing (concentrating and/ordrying) of the fermentation product after fermentation.

Preferably, the fruit ferment further comprises acetate in aconcentration ranging from 0.1 to 60% (w/w). The weight ratio ofpropionate to acetate in such a ferment thereby ranges from 0.5 to 10,preferably from 1.5 to 5.

Also preferably, the fruit ferment further comprises succinate in aconcentration ranging from 0.002 to 1% (w/w). The weight ratio ofpropionate to succinate in such a ferment thereby ranges from 200 to 15,preferably from 150 to 25.

The terms propionate, acetate and succinate as used herein are meant toencompass the acid as well as the salt form. In that regard, typicalsalts are sodium, potassium, ammonium, calcium and magnesium salts, orcombinations thereof.

The fruit ferment further may comprise part or all of the propionicacid-producing bacterial cells and, when used, the lactic acid producingbacterial cells, of the fermentation process.

As a consequence of fermentation, the fruit ferment further has areduced sugar content as compared to the starting liquid fruitpreparation. At least 5% of the amount of sugar present in the startingliquid fruit preparation, preferably at least 10%, more preferably atleast 20%, is converted by fermentation. Sugar in this context refers tohexose mono- and/or disaccharides, including fructose, glucose and/orsucrose.

In one embodiment, the fruit ferment of this aspect is obtainable by themethods as described in the first aspect.

The fruit ferment of this aspect may be a food or beverage as suchand/or may be used as an additive for or an ingredient of a food and/orbeverage.

A further aspect of the invention relates to a composition comprisingthe fruit ferment of the previous aspect.

Such a composition may be an additive composition for food and/orbeverages comprising additional ingredients next to the fruit ferment.Examples of such additional ingredients are lactic acid and/or saltsthereof, acetic acid and/or salts thereof, vinegar, cinnamic acid and/orsalts thereof, vanillin, nisin, sorbic acid and/or salts thereof andfermentation products containing lactic acid and/or salts thereof,acetic acid and/or salts thereof, propionic acid and/or salts thereof,benzoic acid and/or salts thereof, and/or one or more of the followingcomponents: nisin, natamycin, polylysin, bacteriocins.

The fruit ferment or the additive composition comprising the fruitferment as described herein may advantageously be used in food and/orbeverages, for instance to inhibit yeast and/or mould growth in the foodor beverage. Because of the neutral or delicate taste and flavour of thefruit ferment, it does not provide any substantial off-taste and/oroff-flavour to the food or beverage, even when applied in a highconcentration. Typically, the fruit ferment may be added to a food orbeverage to provide a propionate level in the food or beverage in arange of 0.005 to 1% (w/w), preferably in a range of 0.02 to 0.5% (w/w),more preferably in a range of 0.05 to 0.2% (w/w).

In one embodiment, the fruit ferment is advantageously added to afruit-based food and/or beverage, in particular to a fruit-based foodand/or beverage that contains the same type of fruit as the fruit thatis used for preparing the fruit ferment.

In this way, the fruit-based food or beverage obtains novel properties,for instance with regard to taste, flavour, colour, storage stability.Properties like taste, flavour and colour of the fruit ferment arethereby advantageously in line with those of the fruit-based food and/orbeverage. A fruit-based food and/or beverage typically may have a fruitcontent varying from about 3% (w/w), such as in soft drinks, to 100%(w/w), such as in undiluted fruit juices, fruit purées or fruitconcentrates.

Thus, in a further aspect there is provided a method to improve thesensory properties and the storage stability of a fruit-based foodand/or beverage by supplementing the food and/or beverage with a fruitferment as described herein. The fruit ferment thereby preferably has ataste, flavour and/or colour profile that does not substantially deviatefrom the profile of the fruit-based food and/or beverage. For instance,a melon ferment may be added to an apple-based food and/or beverage,whereas a tomato ferment may not. More preferably, the fruit ferment isprepared from the same fruit or fruits as the fruit or fruits includedin the fruit-based food and/or beverage.

The fruit ferment when still containing microbial cells further mayadvantageously be used a probiotic.

EXAMPLE 1

Preparation of Various Fruit Ferments

P. freudenreichii was precultured at a starting pH of 6.5, on a mediumcontaining 15 g/l Difco certified yeast extract and 30 ml/l of a 50%sodium lactate solution. The culture was incubated at 30° C. until itwas fully grown. Lactobacillus paracasei was precultured on MRS medium(de Man et al. 1960, J Appl Bact 23 (130-135), “A Medium for theCultivation of Lactobacilli”), purchased from Oxoid, at a start pH of5.8.

Several cases were tested, with different media compositions. Each caseis listed below in Table 1, wherein the amount of puree or juice perliter fermentation medium is indicated:

Ingredients per liter fermentation medium (adjusted to Case 1 liter withwater) 1 210 g Tomato paste (providing 35.7 g/L sugar) 2 0.5 L watermelon juice (freshly prepared) 3 0.5 L water melon juice (freshlyprepared) 10 g difco yeast extract 4 0.75 L watermelon juice (freshlyprepared) 5 0.25 L apple juice (freshly prepared) 6 0.33 L orange juice(freshly prepared) 7 0.25 L white grape juice (freshly prepared) 8 0.33L pineapple juice (freshly prepared)

The juices in Table 1 were prepared by a standard juice extractor, inthis case a Philips HR1858 juicer. After obtaining the juice, it wasimmediately put into a fermentation reactor without further filtration.

Fermentation reactors were sterilized at 121° C. for 20 minutes. The pHin the reactor was maintained at pH 6.5 with 2.5 M NaOH (100 g/L).Stirring of the media was done at 250 rpm.

At the start of a fermentation, 10% v/v of a culture of lactic acidbacteria and 10% v/v of a culture of propionic acid bacteria were addedto diluted juice.

After fermentation (48-96 hours), solids including biomass were removedusing centrifugation at 7000×g for 20 minutes.

TABLE 1 Acids present in juice after fermentation and solids removal(case 4 not determined) component Case 1 Case 2 Case 3 Case 4 Case 5Case 6 Case 7 Case 8 formic 0.12 0.12 0.19 n.d. 0.04 0.12 0.03 0.07 acidacetic 0.86 0.6 0.8 n.d. 0.52 0.7 0.60 0.71 acid propionic 1.5 1.5 1.4n.d. 1.2 1.1 1.4 1.4 acid ethanol <0.02 0.02 0.04 n.d. <0.01 0.03 <0.01<0.01 butyric <0.01 <0.01 <0.01 n.d. <0.01 <0.01 <0.01 <0.01 acidpyruvic <0.02 <0.02 <0.02 n.d. <0.02 <0.02 <0.02 0.02 acid lactic <0.05<0.05 <0.05 n.d. <0.05 <0.05 <0.05 <0.05 acid 2-hydroxy <0.01 <0.01<0.01 n.d. <0.01 <0.01 <0.01 <0.01 butyric acid oxalic <0.02 <0.01 <0.01n.d. <0.01 <0.01 <0.01 <0.01 acid sorbic <0.01 <0.01 <0.01 n.d. <0.01<0.01 <0.01 <0.01 acid fumaric <0.02 <0.01 <0.01 n.d. <0.01 <0.01 <0.01<0.01 acid succinic 0.10 0.02 0.01 n.d. 0.03 0.02 0.04 0.05 acid benzoic<0.03 <0.03 <0.03 n.d. <0.03 <0.03 <0.03 <0.03 acid maleic <0.02 <0.01<0.01 n.d. <0.01 <0.01 <0.01 <0.01 acid

After solids removal, the ferments were concentrated 8-16 times byrotary vacuum evaporation, operating at 80 mbar. The organic acidcomposition of the concentrated ferments was determined (Table 2):

TABLE 2 Concentration of acids after vacuum evaporation (case 1 notdetermined) component Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7Case 8 formic n.d. 0.51 1.0 1.4 0.24 1.0 0.13 0.37 acid acetic 8.6 3.04.1 9.9 5.4 6.5 4.7 4.3 acid propionic 15   6.8 7.9 22 14 11 12 8.8 acidethanol n.d. <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 butyric n.d.<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 acid pyruvic n.d. <0.02 <0.02<0.02 <0.02 <0.02 <0.02 <0.01 acid lactic n.d. <0.05 <0.05 0.07 <0.05<0.05 <0.05 <0.05 acid 2-hydroxy n.d. <0.01 <0.01 0.01 <0.01 <0.01 <0.01<0.01 butyric acid oxalic n.d. <0.01 <0.01 <0.01 <0.01 0.01 <0.01 0.01acid sorbic n.d. <0.01 <0.01 <0.01 <0.01 0.02 <0.01 0.02 acid fumaricn.d. <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 acid succinic n.d. 0.090.04 1.2 <0.01 0.9 1.0 0.6 acid benzoic n.d. <0.03 <0.03 <0.03 <0.03<0.03 <0.03 <0.03 acid maleic n.d. <0.01 <0.01 <0.01 <0.01 <0.01 <0.01<0.01 acid

In the same manner as described above, four ferments (cases 9, 10, 11and 12) were prepared, but now using a fermentation medium containingper liter of water: 25 g/l Difco yeast extract and 70 g/l sucrose. Afterbiomass removal and concentration, case 9 contained 22% (w/w) propionicacid and case 10 contained 20.4% (w/w) of propionic acid (other acidswere not determined). In case 11, the ferment was evaporated to drynessusing a rotary evaporator at reduced pressure (60-100 mbar). In case 12,the ferment of case 9 was taken and sodium lactic acid was added untilthe ferment contained 25% (w/w) of lactic acid.

After the addition of lactic acid, the ferment contained 13.7% (w/w) ofpropionic acid.

EXAMPLE 2

Effect of Tomato Ferment on Yeasts in Tomato Sauce

The efficacy of a tomato ferment was tested in tomato sauce. Tomatosauce was prepared having a composition according to Table 3:

TABLE 3 Composition of tomato sauce Ingredient Dosage (%) DescriptionSupplier Tomato puree 30.0 Double concentrated 28/30%; Perfektingredients: tomatoes and salt Salt 3.2 Table salt Jozo Sugar 20.0Crystalline sugar CSM Water 45.54 Vinegar 1.26 (=0.36% 100%) (28.5%)TOTAL 100 pH = 3.8 (adjusted with HCl or NaOH), aW = approximately 0.94

A propionate-containing sucrose ferment was prepared based on sucroseand yeast extract, containing 22% propionic acid (case 9 of Example 1).Additionally a concentrated tomato ferment was prepared containing 15%propionic acid (case 1 of Example 1). These ferments were added tobatches of tomato sauce according to Table 4.

The following yeast strains were cultivated on GPY broth (5 g/l yeastextract, 4 g/l glucose, 5 g/l peptone, at a pH of 5-5.5), and added tothe tomato sauce, according to table 4, at an end concentration ofapproximately 1000 cells per gram: Pichia membranaefaciens MUCL 27794,Candida tropicalis MUCL 28180, Zygosaccharomyces rouxii MUCL 30008. Thetomato sauce was incubated at 20° C. with the indicated yeast strain andthe indicated ferment (Table 4). Regularly, the colony forming units(cfu) were determined using MEA plates (20 g/l malt extract, 20 g/lglucose, 1 g/l peptone, 20 g/l agar), incubated at 20° C.

TABLE 4 Test set up of a microbiological experiment with sucrose ferment(case 9) and tomato ferment (case 1) in tomato sauce Tomato Sucroseferment ferment Log cfu added added Log cfu at day Sample Name (% w/w)(% w/w) Microorganism at day 7 80 1 control 0 0 Pichia membranaefaciens3 9.5 1 control 0 0 Candida tropicalis 1.5 <1 1 control 0 0Zygosaccharomyces 4 8.5 rouxii 2 Low tom 1 0 Pichia membranaefaciens <1<1 2 Low tom 1 0 Candida tropicalis <1 <1 3 High tom 3 0 Pichiamembranaefaciens <1 <1 3 High tom 3 0 Candida tropicalis <1 <1 4 Lowprop 0 0.6 Pichia membranaefaciens <1 <1 4 Low prop 0 0.6 Candidatropicalis 1.5 <1 5 High prop 0 1.8 Pichia membranaefaciens <1 <1 5 Highprop 0 1.8 Candida tropicalis <1 <1 6 Med tom 1.7 0 Zygosaccharomyces1.5 <1 rouxii 7 Med prop 0 1 Zygosaccharomyces 1 <1 rouxii

The tomato juice based ferment was as effective as the sucrose basedferment for at least 80 days. It was clear from the results that Candidatropicalis was not able to grow on tomato sauce as the colony formingunits went down in the control experiment in the first 7 days.

EXAMPLE 3

Taste of Melon Ferments Compared to Other Propionate Ferments

This experiment provides a description of the taste of these ferments inapplication and a comparison on taste with other propionate ferments.The comparison is based on the ISO 8587:2006 sensory analysis rankingtest. The taste of the ferments is evaluated in a model drink. Therecipe of this model drink is presented in Table 5.

TABLE 5 recipe of model drink Ingredient Supplier Amount [g] Water949.85 Sucrose Granulated sugar - 40.0 Van Gilse Apple juice concentrateCargill 8.30 Apple flavour Givaudan 55078-DO 0.35 Citric acid Across,M&A-021 1.50

In this model drink the following ferments are compared to each other:

-   -   PSP9 Liquid (see case 10 of Example 1): Concentrated liquid        propionic acid ferment based on sucrose and yeast extract with a        propionic acid content of 20.4% (w/w).    -   PSP9 Powder (see case 11 of Example 1): Powder of the ferment        described above with a propionic acid content of 41% (w/w).    -   Melon ferment with yeast extract: Propionic ferment based on        watermelon juice +10 g/L yeast extract, concentrated.    -   Melon ferment without yeast extract: Propionic ferment based on        watermelon juice, concentrated.    -   PQ (see case 12 of Example 1): Propionic acid ferment based on        sucrose and yeast extract, mixed with lactic acid to a total of        25%.

The ferments were compared at a propionate level of 0.05%. Table 6 showsthe propionate level of each ferment and the amount that should be addedto the model drink to reach a level of 0.05%.

TABLE 6 propionate levels and dosage levels of the ferments FermentPropionate level [%] Dosage level [%] PSP9 Liquid 20.4 0.245 PSP9 Powder41.0 0.122 Melon ferment with 7.9 0.633 yeast extract Melon fermentwithout 6.8 0.735 yeast extract PQ 13.7 0.365

All samples were tasted by 8 trained panellists. The panellists wereasked to rank the samples from least to most different from thereference sample. The reference sample is pure model drink.

Table 7 shows the pH of the samples and the description of the taste.

TABLE 7 pH and description of the taste of samples with differentpropionate ferment Sample pH Description X (reference = model 3.18Fresh, apple drink) PSP9 Liquid 4.21 Less fresh and apple flavour,strong ferment taste PSP9 Powder 4.23 Less fresh and apple flavour,little ferment taste Melon ferment with 4.30 Less fresh and appleflavour, sweet yeast extract Melon ferment without 4.24 Less fresh andapple flavour, sweet, yeast extract little bitter PQ 3.92 Less fresh andapple flavour, sourRanking

The samples were ranked from least to most different from the reference.Table 8 shows the results of the ranking.

TABLE 8 ranking of 5 propionate ferments by 8 panellists Panellist PSP9L PSP9 P Melon + Y Melon − Y PQ n = 8 1 5 3 1 2 4 k = 5 2 5 3 2 1 4 3 54 1 2 3 4 5 4 2 1 3 5 5 2 1 3 4 6 4 1 3 2 5 7 5 3 1 2 4 8 5 1 4 2 3 Sum39 21 15 15 30 SD 0.35 1.19 1.13 0.64 0.71 1 = least different fromreference x, 5 = most different from x

For a panel size of 8 panellists and 5 products to rank the total scoreneeds to be lower than 17 and or higher than 31 to indicate asignificant difference (one-tailed test). It can be seen that both thesamples with melon ferment with and without yeast extract havesignificant less influence on taste compared to the other samples. Thesample with PSP9 Liquid has significant more influence on taste than theother samples. The conclusion is that melon ferments have less influenceon the taste of apple juice model drink than other propionate ferments.The melon ferments have also a different taste profile; they are sweeterthan the other ferments.

EXAMPLE 4

Fermentation of Honey Melon and Apple Juice with Propionibacterium

Preparation of the Preculture

One vial (1 ml) with a −80° C. glycerol stock of Propionibacteriumfreudenreichii ss. shermanii was inoculated in flask of 100 mlcontaining a medium containing 20 g/l of glucose and 15 g/l of yeastextract and incubated at 30° C. for 2 days. The 100 ml of culture wastransferred to a 1 L bottle containing 900 ml of the same medium (20 g/lof glucose and 15 g/l of yeast extract). This bottle was incubated for 1day at 30° C.

Fermentation of Fruit Juice

A 1 L fermenter was prepared with a diluted commercial melon juiceconcentrate. The concentrate was diluted to a sugar concentration ofapproximately 45 g/l, and 1% of yeast extract paste (50% paste fromBiospringer) was added and subsequently the reactor including medium wasautoclaved. The fermentation was started by adding 100 ml of thePropionibacterium preculture. A similar reactor was filled with adiluted commercial apple juice concentrate. The final sugarconcentration was also 45 g/l. Both reactors were kept at 30° C.,stirred at 150 rpm and pH controlled at a pH of 6.5 using an ADI1020controller and 5M NaOH as a base for pH correction. The two reactorswere sampled regularly and the sugar concentration and propionic acidconcentration were measured. The results of these fermentations arepresented in tables 8 and 9. Although the formation of propionic acidwas relatively slow, the glucose was rapidly converted (<48 h) topropionic acid. This suggests that fruits with high glucose content areoptimal in these kinds of fermentations.

TABLE 9 concentrations of acids and sugars in the Honeydew Melon juicefermentation (g/l) Propionic Acetic Succinic Hour acid acid acid GlucoseFructose Sucrose 0 0 0.2 0 13.5 18.1 15.4 24 2.4 1.4 0.4 10.8 16.4 14.548 6.2 3.2 0.8 <1 14.7 14.5 72 7.4 4.1 1.5 <1 11.8 14.1 100 7.9 4.4 2.0<1 9.7 14.1 118 8.2 4.6 2.4 <1 8.0 13.1

TABLE 10 concentrations of acids and sugars in the Apple juicefermentation (g/l) Propionic Acetic Succinic Hour acid acid acid GlucoseFructose Sucrose 0 0 0.1 0 15.3 27.5 2.5 24 1.3 1.0 0.6 12.4 27.5 2.4 485.4 3.1 1.5 <1 24.5 2.4 72 6.3 3.7 2.4 <1 21.0 2.4 100 7.1 4.2 2.8 <118.1 2.4 118 7.9 4.6 3.3 <1 15.7 2.5

The invention claimed is:
 1. A method to prepare a fruit fermentcontaining propionic acid and/or a salt thereof comprising: i) providinga fruit being an edible, seed-associated structure and obtaining aliquid fruit preparation therefrom having a sugar level in a range offrom 1.5 to 10% (w/w) and an initial pH of from 4.5 to 7; and ii)fermenting the liquid fruit preparation with a propionic acid-producingbacterial strain whilst maintaining the pH between 4.5 and 7 duringfermentation by the addition of a base selected from the groupconsisting of sodium hydroxide, potassium hydroxide, magnesiumhydroxide, calcium hydroxide, ammonium hydroxide, calcium carbonate andammonium carbonate; wherein one or more strains of lactic acid producingbacteria are supplementarily used in the fermenting of the liquid fruitpreparation, and the fermentation of the liquid fruit preparation is aprocess that is carried out for a period of from 20 to 120 hours andsaid process is substantially devoid of microorganisms other than saidpropionic acid-producing bacterial strain and said one or more strainsof lactic acid producing bacteria.
 2. The method according to claim 1,wherein the liquid fruit preparation is a fruit juice, a fruit extractor a fruit purée.
 3. The method according to claim 1, wherein the liquidfruit preparation is supplemented with water and/or with additionalcomponents necessary to support and/or improve bacterial fermentation.4. The method according to claim 1, wherein the propionic acid-producingbacterial strain is from the genus Propionibacterium and/or from thespecies Clostridium propionicum, Selenomonas ruminantum and/orBacteroides ruminicola and/or from the genus Veillonella.
 5. The methodaccording to claim 4, wherein the propionic acid-producing bacterialstrain is from the species Propionibacterium freudenreichii,Propionibacterium shermanii and/or Propionibacterium acidi-propionici.6. The method according to claim 1, wherein the method comprisessimultaneously fermenting the liquid fruit preparation with thepropionic acid-producing bacterial strain and one or more strains oflactic acid producing bacteria.
 7. The method according to claim 6,wherein the lactic acid producing bacteria are one or more ofLactobacillus casei, Lactobacillus paracasei, Lactobacillus helveticus,Lactococcus lactis, Lactobacillus acidophilus, Bacillus coagulans,Bacillus smithii, and Bacillus thermoamylovorans.
 8. The methodaccording to claim 1, wherein fermenting the liquid fruit preparation isdone at a pH between 5 and
 7. 9. The method according to claim 1,wherein the lactic acid producing bacteria are one or more ofLactobacillus casei, Lactobacillus paracasei, Lactobacillus helveticus,Lactococcus lactis, Lactobacillus acidophilus, Bacillus coagulans,Bacillus smithii, and Bacillus thermoamylovorans.
 10. The methodaccording to claim 1, wherein the base added to maintain the pH between4.5 and 7 during fermentation is selected from the group consisting ofsodium hydroxide, potassium hydroxide, ammonium hydroxide and ammoniumcarbonate.
 11. A method to prepare a fruit ferment containing propionicacid and/or a salt thereof comprising: i) providing a fruit being anedible, seed-associated structure and obtaining a liquid fruitpreparation therefrom having a sugar level of from 1.5 to 10% (w/w) andan initial pH of from 4.5 to 7; and ii) fermenting the liquid fruitpreparation with a propionic acid-producing bacterial strain whilstsupplementing the liquid fruit preparation with water duringfermentation and maintaining the pH between 4.5 and 7 duringfermentation by the addition of a base selected from sodium hydroxide,potassium hydroxide, magnesium hydroxide, calcium hydroxide, ammoniumhydroxide, calcium carbonate and ammonium carbonate; wherein one or morestrains of lactic acid producing bacteria are supplementarily used inthe fermenting of the liquid fruit preparation, and the fermentation ofthe liquid fruit preparation is a process that is carried out for aperiod of from 20 to 120 hours and said process is substantially devoidof microorganisms other than said propionic acid-producing bacterialstrain and said one or more strains of lactic acid producing bacteria.12. The method according to claim 11, wherein the liquid fruitpreparation is further diluted with water prior to fermentation.
 13. Themethod according to claim 11, wherein prior to and/or duringfermentation the liquid fruit preparation is supplemented withadditional components necessary to support and/or improve bacterialfermentation.
 14. The method according to claim 1, wherein afermentation product obtained from the fermentation of the liquid fruitpreparation is subjected to further processing to obtain the fruitferment containing propionic acid and/or a salt thereof, said furtherprocessing comprising one or more of: a solid/liquid separation step, aconcentration step, a pasteurization or sterilization step, and astandardization step.
 15. The method according to claim 11, wherein afermentation product obtained from the fermentation of the liquid fruitpreparation is subjected to further processing to obtain the fruitferment containing propionic acid and/or a salt thereof, said furtherprocessing comprising one or more of: a solid/liquid separation step, aconcentration step, a pasteurization or sterilization step, and astandardization step.